Characterization of purified myocilin: glaucoma as a protein misfolding disease

纯化肌纤蛋白的表征：青光眼作为一种蛋白质错误折叠疾病

• 批准号: 10357759
• 负责人: Raquel L Lieberman
• 金额: $ 38.89万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10357759
• 关键词: 3-Dimensional; Academia; Affect; Age of Onset; Alleles; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyloidosis; Amyotrophic Lateral Sclerosis; Anatomy; Anterior eyeball segment structure; Aqueous Humor; Benign; Blindness; Cell Death; Cells; Chemicals; Clinic; Comprehension; Data; Databases; Defect; Deuterium; Development; Diagnosis; Disease; Drainage procedure; Endoplasmic Reticulum; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Eye diseases; Fingerprint; Foundations; Functional disorder; Funding; Glaucoma; Glean; Goals; Grant; Growth; High temperature of physical object; Homeostasis; Horns; Hydrogen; Industry; Inherited; Knowledge; Lead; Length; Liquid substance; Mass Spectrum Analysis; Measures; Medical; Medicine; Methods; Missense Mutation; Modality; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Mutagenesis; Mutation; Nerve Degeneration; Nuclear Magnetic Resonance; Open-Angle Glaucoma; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peptides; Physiologic Intraocular Pressure; Physiology; Play; Process; Protein Biochemistry; Proteins; Relaxation; Research Personnel; Retina; Risk Factors; Role; Sampling; Seeds; Structure; Susceptibility Gene; Testing; Therapeutic; Time; TimeLine; Tissues; Toxic effect; Trabecular meshwork structure; Variant; Vertebral column; amyloid formation; biophysical properties; blind; cytotoxic; cytotoxicity; early onset; experimental study; gain of function; genome wide association study; infancy; insight; multidisciplinary; mutant; myocilin; non-Native; novel; novel therapeutics; olfactomedin; patient population; precision medicine; pressure; protein folding; protein misfolding; proteostasis; rare variant; solid state nuclear magnetic resonance; structural biology; superoxide dismutase 1; symptom treatment

Glaucoma, a leading cause of blindness worldwide (70 million patients), is managed medically by treating the symptom of increased intraocular pressure (IOP), but 10% of patients still go blind. IOP is controlled in the anterior segment of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. The TM tissue is diseased in most forms of glaucoma; loss of TM homeostasis leads to elevated IOP. Hereditary open angle glaucoma, affecting ~3 million young patients, is caused by mutations in myocilin, a protein highly expressed in the TM. Since 3/2011, studies funded from R01EY021205 have changed the paradigm for anti-glaucoma therapeutics by laying the molecular foundation for approaches that target the disease process, which are now being pursued in academia and industry. We clarified molecular details of the toxic gain-of-function pathogenic mechanism in which mutant myocilin accumulates in the endoplasmic reticulum (ER) of TM cells, leading to TM cell death and an accelerated timeline for vision loss. Studies from R01EY021205 (a) contributed fundamental knowledge of myocilin structure, (b) discovered a counter-productive interaction between myocilin and the ER-resident Hsp90 chaperone Grp94, and (c) characterized myocilin misfolding as amyloid. Wild-type and many different myocilin variants harbor a misfolding propensity; thus, proteostasis issues identified in familial myocilin- associated glaucoma are likely at play in many more patients. Amyloid formation by myocilin places glaucoma alongside more well-studied amyloid diseases like Alzheimer and SOD-1 dependent amyotrophic lateral sclerosis, yet our comprehension of the role of amyloid in glaucoma is in its infancy. Our current objective is to better understand molecular aspects of myocilin fibrilization, focused on the relevant olfactomedin (OLF) domain. Our multidisciplinary team will (a) clarify initiation of aggregation by studying solution structures of wild-type and selected OLF variants, as well as corresponding multi-length scale dynamics, using hydrogen-deuterium exchange mass spectrometry and nuclear magnetic resonance (NMR) structure and relaxation methods (Wade Van Horn, Co-I) (b) compare the end-point structures of selected OLF aggregates to known amyloids by solid state NMR (Anant Paravastu, Co- I) and evaluate cytotoxicity of intermediate aggregates and (c) evaluate common allele full-length myocilin variants for experimental hallmarks of pathogenicity. The expected outcome is a better understanding of the myocilin misfolding process at the molecular level, including molecular determinants of pathogenicity, to enable novel modalities for studying, diagnosing, and treating myocilin-associated glaucoma. More broadly, continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but will also extend our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases.

青光眼是世界范围内导致失明的主要原因之一(7000万患者)，通过治疗进行医学治疗。 眼压升高的症状，但仍有10%的患者失明。眼压控制在 眼前段，其中包含小梁网络(TM)细胞外基质，解剖 房水引流通道。TM组织在大多数形式的青光眼中都有病变； TM动态平衡导致眼压升高。遗传性开角型青光眼，影响约300万年轻患者， 是由TM中高度表达的一种蛋白质myoclin的突变引起的。自2011年3月起，资助的研究 R01EY021205通过奠定分子基础改变了抗青光眼治疗的范式 针对疾病过程的方法，目前学术界和工业界正在探索这一方法。 我们阐明了毒性功能获得致病机制的分子细节 霉菌素积聚在TM细胞的内质网(ER)中，导致TM细胞死亡和AN 视力丧失的加速时间线。来自R01EY021205(A)的研究贡献了基础知识 Myocin结构，(B)发现myoclin和内质网驻留之间的反生产相互作用 HSP90分子伴侣Grp94，和(C)将myocin错误折叠鉴定为淀粉样蛋白。野性的和许多不同的 肌球蛋白变异体有错误折叠的倾向；因此，在家族性肌球蛋白中发现的蛋白平衡问题-- 相关的青光眼可能在更多的患者中起作用。 由霉菌蛋白形成的淀粉样蛋白使青光眼与更多研究较好的淀粉样蛋白疾病并存，如 阿尔茨海默病和SOD-1依赖的肌萎缩侧索硬化症，但我们对淀粉样蛋白在 青光眼还处于婴儿期。我们目前的目标是更好地了解肌球蛋白的分子方面 纤维化，侧重于相关的嗅觉素(OLF)领域。我们的多学科团队将(A)澄清 通过研究野生型和选定的OLF变体的溶液结构以及 相应的多长度标度动力学，使用氢-氚交换质谱仪和 核磁共振结构和松弛方法(Wade Van Horn，Co-I)(B)比较 固体核磁共振研究已知淀粉样蛋白的OLF聚集体的端点结构(Anant Paravastu，Co- I)和评价中间聚集体的细胞毒性以及(C)评价常见等位基因全长myoclin 致病性实验特征的变种。预期的结果是更好地理解 霉菌素在分子水平上的错误折叠过程，包括致病的分子决定因素，使 研究、诊断和治疗肌球蛋白相关性青光眼的新方法。更广泛地说，继续 研究肌球蛋白的结构和功能不仅有助于我们理解青光眼及其作用 在TM中，但也将扩展我们对许多其他OLF领域的理解，这些领域涉及 在生理学和疾病方面广泛存在。